This website uses cookies to deliver some of our products and services as well as for analytics and to provide you a more personalized experience. Click here to learn more. By continuing to use this site, you agree to our use of cookies. We've also updated our Privacy Notice. Click here to see what's new.

This website uses cookies to deliver some of our products and services as well as for analytics and to provide you a more personalized experience. Click here to learn more. By continuing to use this site, you agree to our use of cookies. We've also updated our Privacy Notice. Click here to see what's new.

About Optics & Photonics TopicsOSA Publishing developed the Optics and Photonics Topics to help organize its diverse content more accurately by topic area. This topic browser contains over 2400 terms and is organized in a three-level hierarchy. Read more.

Topics can be refined further in the search results. The Topic facet will reveal the high-level topics associated with the articles returned in the search results.

Abstract

In light sheet fluorescence microscopy optical sectioning is achieved by illuminating the sample orthogonally to the detection pathway with a thin, focused sheet of light. However, light scattering within the sample often deteriorates the optical sectioning effect. Here, we demonstrate that contrast and degree of confocality can greatly be increased by combining scanned light sheet fluorescence excitation and confocal slit detection. A high frame rate was achieved by using the “rolling shutter” of a scientific CMOS camera as a slit detector. Synchronizing the “rolling shutter” with the scanned illumination beam results in confocal line detection. Acquiring image data with selective plane illumination minimizes photo-damage while simultaneously enhancing contrast, optical sectioning and signal-to-noise ratio. Thus the imaging principle presented here merges the benefits of scanned light sheet microscopy and line-scanning confocal imaging.

Figures (7)

Fig. 1 Illumination beam path. (A) Top view. The excitation beam exits a single mode fiber and is guided by mirrors onto a scanning mirror. The scanning position is imaged by a relay objective into the back focal plane of the illumination objective lens, which is located inside the objective housing. The relay lens assembly is composed of two lens systems with overlapping focal points. It also serves to expand the illumination beam. (B) Side view of the illumination optics.

Fig. 2 Rolling shutter mode. An activated row was marked in red, and a read-out row was marked in yellow. The band of pixel rows in between is exposed to light simultaneously. This band moves from the top to the bottom of the sensor. Its width is defined by the single row exposure time, and can be adjusted from a minimum of one line up to the whole chip.

Fig. 4 Images of beads in an agarose gel demonstrating the contrast difference between global shutter and rolling shutter image acquisition. (A) Image taken in global shutter mode, (B) in rolling shutter mode with a slit width of 128 rows, (C) in rolling shutter mode with a slit width of 32 rows and (D) in rolling shutter mode with a slit width of 2 rows. The image acquisition time was 22 ms in each image. Scale bar, 50 μm.

Fig. 5 3D reconstruction of a 120x120x50 μm3volume of beads fixed in agarose. The sample volume was imaged in (A) global shutter mode and (B) with a rolling shutter of two lines. The reduction of background in the reconstructed rolling shutter data is evident. The rolling shutter data were averaged 40x to yield an SNR comparable to the global shutter images.

Fig. 6 SNR and contrast. (A) SNR for different rolling shutter sizes in units of the illumination beam diameter, and for global shutter. (B) Contrast as a function of rolling shutter size, and for global shutter. The rolling shutter sizes corresponding to each point are 2, 4, 8, 16, 32, 64, 128 and 256 pixels, starting from the left.

Fig. 7 Images of polytene chromosomes in C. tentans salivary gland cell nucleus. (A) Image taken in global shutter mode. The beam was swept once across the sample. (B) Nucleus imaged with a rolling shutter width corresponding to the illumination beam diameter. The bands are crisper and the background in dark areas of the sample is lower as compared to (A). Scale bar, 30 μm. (C) Normalized intensity along the blue and red line in (A) and (B), respectively.